Alpha1-proteinase inhibitor (abbreviated herein as A1PI; also known as alpha-1 protease inhibitor, alpha-1 PI, A1PI, α-1 PI, α1PI, alpha-1 trypsin inhibitor, alpha1 antitrypsin, alpha-1 antitrypsin, alpha1AT, A1A, and A1AT, AAT, inter alia), is the major serine protease inhibitor (serpin) in humans. A1PI is expressed as a 418 amino acid protein with residues 1-24 being a signal peptide. The mature protein, consisting of residues 25-418, is a single chain glycoprotein having a molecular weight of about 51 kD. See FIG. 1. While A1PI does not contain any disulfide bonds, the protein is highly structured, with 80% of the amino acids residing in eight well-defined α-helices and three large β-sheets. Three asparagine-linked carbohydrates are found on Asn 70, Asn 107, and Asn 271 (numbered as in the full-length protein). This gives rise to multiple A1PI isoforms, having isoelectric points in the range of 4.0 to 5.0. The glycan monosaccharides include N-acetylglucosamine, mannose, galactose, fucose, and sialic acid.
Normal plasma concentrations of A1PI range from 1.3 to 3.5 mg/mL. A1PI functions by protecting cells from proteases involved in clotting and inflammation. A1PI inhibits trypsin, chymotrypsin, various forms of elastases, skin collagenase, renin, urokinase, and proteases of polymorphonuclear lymphocytes, among others. A1PI serves as a pseudo-substrate for these proteases, which attack the reactive center loop of the A1PI molecule (residues Gly 368-Lys 392) by cleaving the bond between residues Met 358-Ser 359 forming an A1PI-protease complex. This complex is rapidly removed from the blood circulation. One of the endogenous roles of A1PI is to regulate the activity of neutrophil elastase, which breaks down foreign proteins and injures native tissue present in the lung. In the absence of sufficient quantities of A1PI, the elastase breaks down lung tissue, which over time results in chronic lung tissue damage and emphysema.
A1PI is often purified from blood plasma. See, e.g., U.S. Pat. Nos. 6,284,874; 6,462,180; 6,093,804; 7,879,800; and WO 1998/000154; WO 2002/048176; WO 2010/009388, for example. In addition, recombinant A1PI (recA1PI) can be expressed and purified from a variety of sources. See, e.g., U.S. Pat. Nos. 4,931,373 and 5,134,119; U.S. Patent Application Publications Nos. US 2004/0124143 and US 2007/0218535; PCT Publication Nos. WO 2005/047323 and WO 2010/127939; and Archibald et al., Proc. Natl. Acad. Sci. USA 87:5178-5182 (1990); Wright et al., Nat. Biotechnology 9: 830 (1991).
Methods for expressing and purifying human recombinant, cell culture derived A1PI for therapeutic use are described herein. Following purification, the A1PI solution had a yellow or amber color that may be objectionable to clinicians and/or patients. Methods for diminishing the coloration are also described herein.